New insights are emerging into the role of CD1d and NKT cells in the host response to many microbial infections. In some of these infections, microbial lipid antigens are presented by CD1d and recognized by the semi-invariant T cell receptor (TCR) expressed by invariant (i) NKT cells. This is called direct, antigen-driven iNKT cell activation. However, only a few (3-4) such cognate lipid antigens from all of the major pathogens have been found and some microbes, like viruses, that can activate iNKT cells, do not encode any lipid antigens. This led to the identification of another mechanism of iNKT cell activation by microbes that is not dependent on recognition of microbial lipid antigens. Instead, microbial TLR agonists stimulate antigen-presenting cells (APC) to secrete cytokines like IL-12 that synergize with a weaker TCR stimulation provided by CD1d bound self-lipid antigens on the APC. These combined signals evoke strong iNKT cell activation, in the absence of iNKT cell TCR recognition of microbial lipid antigens directly, and thus is called indirect, cytokine-driven iNKT cell activation. Fungi cause important opportunistic infections and allergic pathology in humans. We have found that the fungal pathogen, Aspergillus fumigatus, efficiently activates iNKT cells by the indirect, cytokine-driven mechanism. However, instead of requiring TLR signaling, fungal activation of iNKT cells is dependent on the characteristic fungal 2-glucans and the signaling C-type lectin receptor, Dectin-1. Here we will define this mechanism of iNKT cell activation in vitro (Aim 1) and in vivo (Aim 2) and the role of iNKT cells in control of invasive fungal infection in vivo in mouse models (Aim 3). Finally, we will show the significance of the indirect, cytokine-driven iNKT cell activation mechanism in vivo for representative gram (-) and gram (+) bacterial microbes, including organisms known to have cognate CD1d presented microbial lipid antigens recognized by the iNKT TCR. Together, these studies will define the mechanism of a new pathway of TLR independent iNKT cell activation, its role in control of fungal infection and overall significance of indirect versus direct activation of iNKT cells in vivo.